FRMD4A gene molecular marker related to large white pig nipple number character, detection method and application

Abstract

The invention belongs to the technical field of molecular biology, and relates to a large white pig nipple number character related FRMD4A gene SNP (Single Nucleotide Polymorphism) marker as well as a detection method and application thereof. The SNP marker is located on a nucleotide sequence of an FRMD4A gene on a pig chromosome 10, a site of the SNP marker is a g.48019215 nucleotide site on the pig chromosome 10 of a reference sequence of an international pig genome version 11.1, the SNP marker has C / T polymorphism, and the SNP marker is remarkably related to the nipple number character. The SNP marker provided by the invention is related to pig nipple number characters, pig strains with multiple nipples can be screened by identifying the SNP marker, and the obtained strains with multiple nipples and high reproductive capacity have important economic benefits and social values.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and relates to a FRMD4A gene molecular marker related to the teat number trait of Large White pigs, a detection method and an application thereof. Background Art

[0002] Large White pigs, as a commercial pig breed widely raised around the world, occupy an important position in modern animal husbandry with their excellent growth performance and reproductive capacity. The number of nipples is an important reproductive trait of pigs, which directly affects the lactation ability of sows and the survival rate of piglets. The more nipples a sow has, the larger the effective lactation space it can provide, which is essential for providing sufficient nutrition during lactation and promoting the healthy development of piglets. With the significant improvement in litter size in recent years, the number of piglets in a litter is sometimes greater than the number of functional nipples of a sow, which limits piglet production. How to improve the lactation ability of sows, increase the lactation space of sows, and essentially improve the nutritional intake of piglets and improve the survival rate of piglets has become a top priority in the current development of animal husbandry. The development and use of new molecular breeding markers to increase the number of nipples has attracted much attention.

[0003] In commercial pig breeding, the number of nipples is an important indicator for evaluating the reproductive performance of sows. Sows with more nipples are usually selected for breeding to improve the reproductive efficiency of the overall group. The number of nipples is regulated by multiple genes on different chromosomes, with a heritability of about 0.32 to 0.39, which is a medium heritability trait. Traditional breeding methods based on pedigree and phenotypic records are costly and have poor improvement effects on reproductive traits. The FRMD4A (FERMdomaincontaining4A) gene plays an important role in mammary gland development. Genome-wide association analysis (GWAS) showed that FRMD4A is a key candidate gene for regulating the number of nipples in Large White pigs. The development of the mammary gland and nipples depends on the polarization of epithelial cells. During the formation of the nipple, epithelial cells polarize to form different membrane regions, allowing cells to differentiate and perform specific functions. The differentiation of the apical membrane and basement membrane helps the mammary ducts form tubular structures at the nipple and ensures the functional characteristics of the nipple tissue. Arf6 is a core player in actin cytoskeleton dynamics and membrane trafficking. It is activated when the original "spotted" adherens junctions are formed and is necessary for establishing epithelial polarity. Studies have found that FRMD4A regulates the activation of Arf6 and affects the epithelial polarization process by connecting specific guanine nucleotide exchange factors (GEFs) of the cytokines family to Par-3 proteins. The lack of the FRMD4A gene will hinder the development of nipples. Therefore, it is of great significance to identify the FRMD4A genotype and apply it to marker-assisted selection to breed new strains with more nipples, improve the lactation ability of sows, and enhance the nutritional level of piglets. Summary of the invention

[0004] The purpose of the present invention is to provide a FRMD4A gene molecular marker related to the multiple nipple number trait and its breeding application in view of the shortcomings of the existing technology and the difficulty in predicting and breeding pigs with the multiple nipple number trait in advance.

[0005] Another object of the present invention is to provide primers and detection methods for detecting the above-mentioned molecular markers and related applications.

[0006] The technical solution provided by the present invention is: a FRMD4A gene molecular marker related to the multiple nipple number trait, the SNP marker is located on the FRMD4A gene nucleotide sequence from 47384867 to 48048948 on pig chromosome 10, the SNP marker site is the g.48019215 nucleotide site on pig chromosome 10 of the international pig genome version 11.1 reference sequence, and has a C / T polymorphism, the SNP marker is significantly correlated with the nipple number trait, and the number of nipples of pigs with CC genotype at this site is significantly higher than that of pigs with TT genotype.

[0007] Preferably, the molecular marker sequence is as shown in SEQ ID NO: 1, and the SNP marker site is located at the 301st position where the C / T polymorphism exists.

[0008] At the same time, the present invention also provides an amplification primer for amplifying the FRMD4A gene molecular marker related to the Large White Pig teat number trait, or for detecting the Large White Pig teat number trait, which is designed based on the nucleotide sequence containing the SNP marker.

[0009] Preferably, the upstream primer sequence of the primer is shown as SEQ ID NO: 2, and the downstream primer sequence is shown as SEQ ID NO: 3.

[0010] The present invention also provides a method for detecting the FRMD4A gene molecular marker associated with the Large White Pig teat number trait or detecting the Large White Pig teat number trait, comprising the following steps: A sequence containing the SNP marker in the pig genome is amplified by PCR, and the amplified product is sequenced to determine the C / T polymorphism of the site.

[0011] The method specifically comprises the following steps: (1) Taking large white pig ear tissue samples and extracting total DNA; (2) using the extracted porcine genomic DNA as a template and performing PCR amplification using the primers described in claim 3 or 4; (3) The amplified product was sequenced, the sequencing results were analyzed, and the C / T polymorphism at position 301 of SEQ ID NO: 1 was determined.

[0012] Furthermore, in the method, the PCR amplification reaction system in step (2) is: 1.0 μL of DNA template, 1.0 μL of primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, 0.125 μL of Taq enzyme reagent, and 17.38 μL of double distilled water; wherein the concentration of the DNA template is 50 ng / μL, the concentration of the primers is 10 mol / L, and the Taq enzyme is a reagent from Dalian TaKaRa Biotechnology Co., Ltd.; the reaction procedure for PCR amplification is: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 10 s; annealing at 60°C for 30 s, extension at 72°C for 30 s, 34 cycles; extension at 72°C for 5 min.

[0013] The present invention also provides the application of the SNP marker, the amplification primer, or the method in detecting the teat number trait of Large White pigs or screening pig strains with a large teat number.

[0014] In the application, the screening of high-nipple-number pig strains is achieved by the following method: detecting the genotype of the piglet at the nucleotide site g.48019215, and selecting CC-type individuals at the nucleotide site g.48019215 as high-fertility breeding pigs with high nipples.

[0015] The present invention also provides a method for breeding high-fertility sows with a large number of nipples. The method comprises obtaining offspring piglets by self-pollination or hybridization, detecting the genotype of the nucleotide site g.48019215 of the piglets by the method described in any one of claims 5 to 7, and selecting CC-type individuals at the nucleotide site g.48019215 as high-fertility sows with a large number of nipples.

[0016] The present invention has the following beneficial effects: The FRMD4A gene molecular marker provided by the present invention is related to the multiple nipple number trait. Therefore, pig strains with multiple nipples can be screened by identifying the SNP marker. The obtained multiple nipple number pig strains show higher lactation performance and fertility, and have important economic benefits and social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of chromosome-level significant SNP markers in genome-wide association analysis of nipple number trait. The information of 18 autosomes and X chromosome of pigs is marked on the x-axis.

[0018] Figure 2 The electrophoresis diagram is the result of amplifying the FRMD4A gene using the primers of the present invention.

[0019] Figure 3 This is the peak diagram of DNA sequencing results for different genotypes of the molecular marker site of the FRMD4A gene. DETAILED DESCRIPTION

[0020] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0021] Example 1 1. Source of experimental animals Collected from 200 Large White pigs of Shaanxi Huamu Xingnong Breeding Pig Technology Co., Ltd.

[0022] 2. Phenotypic determination When the sows reach 120 days of age, effective nipples are counted and non-functional nipples, such as blind nipples, inverted nipples, and superfluous nipples, are removed.

[0023] 3. Genomic DNA Extraction Pig ear tissue samples were collected, placed in a centrifuge tube filled with 70% alcohol, and stored in a -20°C refrigerator for later use.

[0024] Use the animal genomic DNA extraction kit to extract genomic DNA. The required reagents include: Proteinase K (Beijing Solebow Technology Co., Ltd.) Animal genomic DNA extraction kit: Sangon Biotech (Shanghai) Co., Ltd. DNAMarker (DL1000): Shanghai Pudi Biotechnology Co., Ltd. 10×LoadingBuffer: Bio-Tech (Beijing) Co., Ltd. Agarose: Sangon Biotech (Shanghai) Co., Ltd. TBE (10×) solution: Shanghai Bio-Tech Biotechnology Co., Ltd.

[0025] The specific steps are as follows: (1) Take 25 mg of left and right ear tissues and place them in a centrifuge tube. Add them to a 1.5 ml centrifuge tube and add 400 µl of Buffer Digestion. Mix by vortexing. Incubate in a 65°C water bath for 1 h until the cells are completely lysed.

[0026] (2) Add 20 μL of Proteinase K solution, shake to mix, and place in a 56°C water bath to digest overnight.

[0027] (3) Add 200 µl of Buffer PA, mix thoroughly by inversion, and place in a -20°C refrigerator for 5 min.

[0028] (4) Centrifuge at 10,000 rpm for 5 min at room temperature and transfer the supernatant (500-550 µl) to a new 1.5 ml centrifuge tube.

[0029] (5) Add an equal volume of isopropanol, invert 5 to 8 times to mix thoroughly, and leave at room temperature for 2 to 3 minutes. Centrifuge at 10,000 rpm for 5 minutes at room temperature and discard the supernatant.

[0030] (6) Add 1 ml of 75% ethanol, invert and rinse for 1-3 minutes, centrifuge at 10,000 rpm for 2 minutes, and discard the supernatant. (7) Repeat step 6.

[0031] (7) Open the lid and invert at room temperature for 5 to 10 minutes until the remaining ethanol evaporates completely.

[0032] (8) Dissolve the obtained DNA in 50~100µl TE Buffer. The extracted DNA can be used for the next experiment immediately or stored at -20°C.

[0033] (9) Determine the concentration. After checking the mass and concentration using a spectrophotometer, dilute the sample to 50 ng / μL and store at -20°C for later use.

[0034] 4. Pig genome 60K SNP chip genotyping and genome-wide association analysis The whole genome genotype of pigs was determined according to the company's standard process. The "Zhongxin No. 1" pig 50KSNP chip was used to detect the SNP genotype of individual pigs with nipple number determination records, and all SNP marker detection results were quality controlled. Three genome-level significant SNPs were detected using whole genome association analysis and concentrated on pig chromosome 10 ( Figure 1 ). By comparing with the international pig genome version 11.1 reference sequence, one of the SNPs significantly associated with nipple number ( P =4.63E-09) is located in the coding region of the FRMD4A gene. According to existing literature and functional annotations, the FRMD4A gene is essential for epithelial cell polarization. The lack of this gene will lead to blocked nipple development. SNP variation in the FRMD4A gene may regulate and affect the phenotype of pig nipple number.

[0035] Example 2 This example verifies the SNP site g.48019215C / T on FRMD4A that is significantly associated with the teat number trait obtained in Example 1 in a large white pig population. SEQ ID NO: 1 is the product sequence after PCR amplification of the designed primers SEQ ID NO: 2 and SEQ ID NO: 3, and the SNP site significantly associated with the teat number is the 301st mutation in the sequence.

[0036] 1. Extraction of pig genomic DNA The ear tissue samples of the sows with nipple number trait records were collected, placed in a centrifuge tube filled with 70% alcohol, and stored in a -20°C refrigerator for later use. The genomic DNA of the pig ear tissue was extracted using the above method, and after quality and concentration testing, the concentration was diluted to 50 ng / μL and stored at -20°C for later use.

[0037] 2. PCR amplification and sequencing of target fragments Using the extracted pig DNA as a template, PCR amplification was performed according to the designed primers: the PCR amplification reaction system was: 1.0 μL of DNA template, 1.0 μL of primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, 0.125 μL of Taq enzyme reagent, and 17.38 μL of double distilled water; the PCR amplification reaction procedure was: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 10 s; annealing at 60°C for 30 s, extension at 72°C for 30 s, 30 cycles; extension at 72°C for 5 min.

[0038] The PCR product was detected by electrophoresis in 1.2% agarose gel. The size of the amplified target fragment was 412 bp. Figure 2 The remaining amplified products were sequenced, and the sequencing results were compared and analyzed with the pig FRMD4A gene fragment sequence in GenBank using DNAMAN software, and the g.48019215C / T genotype was interpreted. The chi-square test module of SAS9.4 software was used to perform single marker association analysis between SNP genotype and teat number trait.

[0039] Table 1 shows the effect of the g.48019215C / T mutation site on the teat number trait in the resource population. As shown in Table 1, compared with the TT genotype individuals at the SNP site (g.48019215C / T), the CC genotype individuals had a significant difference in teat number (P<0.05). This shows that in the selection of sows, the CC genotype individuals at the g.48019215C / T site can gradually increase the number of sow teats and improve the lactation ability and fertility of sows.

[0040] Table 1. Association analysis between SNP locus (g.48019215C / T) and nipple number trait .

Claims

1. A FRMD4A gene molecular marker associated with the number of teats in Large White pigs, characterized in that: The SNP marker is located on the FRMD4A gene nucleotide sequence at positions 47384867 to 48048948 on pig chromosome 10. The SNP marker site is the g.48019215 nucleotide site on pig chromosome 10 of the international pig genome version 11.1 reference sequence, and has a C / T polymorphism, wherein the number of nipples of the CC genotype at the site is significantly higher than that of the TT genotype pig.

2. The FRMD4A gene molecular marker associated with the number of teats in Large White pigs according to claim 1, characterized in that: The molecular marker sequence is shown as SEQ ID NO: 1, and the SNP marker site is located at the 301st position where C / T polymorphism exists.

3. A primer for amplifying the FRMD4A gene molecular marker associated with the Large White Pig teat number trait as claimed in claim 1 or 2, or for detecting the Large White Pig teat number trait, characterized in that: It is designed based on the nucleotide sequence containing the SNP marker as claimed in claim 1.

4. The amplification primer according to claim 3, characterized in that The upstream primer sequence of the primer is shown as SEQ ID NO: 2, and the downstream primer sequence is shown as SEQ ID NO:

3.

5. A method for detecting the FRMD4A gene molecular marker associated with the Large White Pig teat number trait as claimed in claim 1 or 2 or detecting the Large White Pig teat number trait, characterized in that: The steps include: A sequence containing the SNP marker in the pig genome is amplified by PCR, and the amplified product is sequenced to determine the C / T polymorphism of the site.

6. The method according to claim 5, characterized in that The following steps are involved: (1) Taking large white pig ear tissue samples and extracting total DNA; (2) using the extracted porcine genomic DNA as a template and performing PCR amplification using the primers described in claim 3 or 4; (3) The amplified product was sequenced, the sequencing results were analyzed, and the C / T polymorphism at position 301 of SEQ ID NO: 1 was determined.

7. The method according to claim 6, characterized in that The PCR amplification reaction system described in step (2) is: 1.0 μL of DNA template, 1.0 μL of primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, 0.125 μL of Taq enzyme reagent, and 17.38 μL of double distilled water; wherein the concentration of the DNA template is 50 ng / μL, and the concentration of the primers is 10 mol / L; the reaction procedure of PCR amplification is: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 10 s; annealing at 60°C for 30 s, extension at 72°C for 30 s, 34 cycles; extension at 72°C for 5 min.

8. Use of the SNP marker according to claim 1 or 2, the amplification primer according to claim 3 or 4, or the method according to any one of claims 5 to 7 in detecting the teat number trait of Large White pigs or screening high teat number pig strains.

9. The use according to claim 8, characterized in that The screening of pig strains with a high nipple number is achieved by the following method: detecting the genotype of the nucleotide site g.48019215 of the piglets, and selecting CC-type individuals with the nucleotide site g.48019215 as high-fertility breeding pigs with a high nipple number.

10. A method for breeding high-fertility pigs with a large number of nipples, characterized in that: Obtain offspring piglets through self-pollination or hybridization, detect the genotype of the nucleotide site g.48019215 of the piglets using the method described in any one of claims 5 to 7, and select CC type individuals at the nucleotide site g.48019215 as high-fertility piglets with multiple nipples.

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